Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

BACKGROUND: Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. METHODS: Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. RESULTS: Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. CONCLUSIONS: The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

High temperatures and low humidity promote the occurrence of microsporidians (Microsporidia) in mosquitoes (Culicidae).

BACKGROUND: In the context of climate change, a growing concern is that vector-pathogen or host-parasite interactions may be correlated with climatic factors, especially increasing temperatures. In the present study, we used a mosquito-microsporidian model to determine the impact of environmental factors such as temperature, humidity, wind and rainfall on the occurrence rates of opportunistic obligate microparasites (Microsporidia) in hosts from a family that includes important disease vectors (Culicidae). METHODS: In our study, 3000 adult mosquitoes collected from the field over 3 years were analysed. Mosquitoes and microsporidia were identified using PCR and sequencing of the hypervariable V5 region of the small subunit ribosomal RNA gene and a shortened fragment of the cytochrome c oxidase subunit I gene, respectively. RESULTS: DNA metabarcoding was used to identify nine mosquito species, all of which were hosts of 12 microsporidian species. The prevalence of microsporidian DNA across all mosquito samples was 34.6%. Microsporidian prevalence in mosquitoes was more frequent during warm months (> 19 °C; humidity < 65%), as was the co-occurrence of two or three microsporidian species in a single host individual. During warm months, microsporidian occurrence was noted 1.6-fold more often than during the cold periods. Among the microsporidians found in the mosquitoes, five (representing the genera Enterocytospora, Vairimorpha and Microsporidium) were positively correlated with an increase in temperature, whereas one (Hazardia sp.) was significantly correlated with a decrease in temperature. Threefold more microsporidian co-occurrences were recorded in the warm months than in the cold months. CONCLUSIONS: These results suggest that the susceptibility of mosquitoes to parasite occurrence is primarily determined by environmental conditions, such as, for example, temperatures > 19 °C and humidity not exceeding 62%. Collectively, our data provide a better understanding of the effects of the environment on microsporidian-mosquito interactions.

Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Arboviruses can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important arboviruses, including dengue virus (DENV), Zika virus (ZIKV), Chikungunya (CHIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of DENV (as a model system for mosquito-borne viruses more generally) that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP)-the time taken for DENV virus to be transmissible after infection-is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicate that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection.

Towards modelling tick-virus interactions using the weakly pathogenic Sindbis virus: Evidence that ticks are competent vectors.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

Biased virus transmission following sequential coinfection of Aedes aegypti with dengue and Zika viruses.

BACKGROUND: Mosquito-borne arboviruses are expanding their territory and elevating their infection prevalence due to the rapid climate change, urbanization, and increased international travel and global trade. Various significant arboviruses, including the dengue virus, Zika virus, Chikungunya virus, and yellow fever virus, are all reliant on the same primary vector, Aedes aegypti. Consequently, the occurrence of arbovirus coinfection in mosquitoes is anticipated. Arbovirus coinfection in mosquitoes has two patterns: simultaneous and sequential. Numerous studies have demonstrated that simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses. However, the viruses' interplay within a mosquito after the sequential coinfection seems intricated and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We conducted experiments aimed at examining the phenomenon of arbovirus sequential coinfection in both mosquito cell line (C6/36) and A. aegypti, specifically focusing on dengue virus (DENV, serotype 2) and Zika virus (ZIKV). We firstly observed that DENV and ZIKV can sequentially infect mosquito C6/36 cell line, but the replication level of the subsequently infected ZIKV was significantly suppressed. Similarly, A. aegypti mosquitoes can be sequentially coinfected by these two arboviruses, regardless of the order of virus exposure. However, the replication, dissemination, and the transmission potential of the secondary virus were significantly inhibited. We preliminarily explored the underlying mechanisms, revealing that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, leading to reduced susceptibility to the secondary arbovirus infections. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. However, it is important to note that more experimental investigations are needed to refine and expand upon this conclusion.

Anopheles aquatic development kinetic and adults' longevity through different seasons in laboratory and semi-field conditions in Burkina Faso.

ABSTRACT: BACKGROUND: Anopheles mosquitoes are ectothermic and involved in numerous pathogen transmissions. Their life history traits are influenced by several environmental factors such as temperature, relative humidity and photoperiodicity. Despite extensive investigations of these environmental conditions on vector population ecology, their impact on the different life stages of Anopheles at different seasons in the year remains poorly explored. This study reports the potential impact of these abiotic factors on the immature and adult stages of Anopheles gambiae sensu lato during different seasons. METHODS: Environmental conditions were simulated in the laboratory using incubators to mimic the environmental conditions of two important periods of the year in Burkina Faso: the peak of rainy season (August) and the onset of dry season (December). Eggs from wild An. coluzzii and An. gambiae s.l. were reared separately under each environmental condition. For Anopheles coluzzii or An. gambiae s.l., eggs were equally divided into two groups assigned to the two experimental conditions. Four replicates were carried out for this experiment. Then, egg hatching rate, pupation rate, larval development time, larva-to-pupae development time, adult emergence dynamics and longevity of Anopheles were evaluated. Also, pupae-to-adult development time from wild L3 and L4 Anopheles larvae was estimated under semi-field conditions in December. RESULTS: A better egg hatching rate was recorded overall with conditions mimicking the onset of the dry season compared to the peak of the rainy season. Larval development time and longevity of An. gambiae s.l. female were significantly longer at the onset of the dry season compared than at the peak of the rainy season. Adult emergence was spread over 48 and 96 h at the peak of the rainy season and onset of dry season conditions respectively. This 96h duration in the controlled conditions of December was also observed in the semi-field conditions in December. CONCLUSIONS: The impact of temperature and relative humidity on immature stages and longevity of An. gambiae s.l. adult females differed under both conditions. These findings contribute to a better understanding of vector population dynamics throughout different seasons of the year and may facilitate tailoring of control strategies.

The effect of physical barriers under a raised house on mosquito entry: an experimental study in rural Gambia.

BACKGROUND: Anopheles gambiae, the major malaria mosquito in sub-Saharan Africa, feed largely indoors at night. Raising a house off the ground with no barriers underneath reduces mosquito-house entry. This experiment tested whether walling off the space under an elevated hut affects mosquito-hut entry. METHODS: Four inhabited experimental huts, each of which could be moved up and down, were used in rural Gambia. Nightly collections of mosquitoes were made using light traps and temperature and carbon dioxide levels monitored indoors and outdoors using loggers. Each night, a reference hut was kept at ground level and three huts raised 2 m above the ground; with the space under the hut left open, walled with air-permeable walls or solid walls. Treatments were rotated every four nights using a randomized block design. The experiment was conducted for 32 nights. Primary measurements were mosquito numbers and indoor temperature in each hut. RESULTS: A total of 1,259 female Anopheles gambiae sensu lato were collected in the hut at ground level, 655 in the hut with an open ground floor, 981 in the hut with air-permeable walls underneath and 873 in the hut with solid walls underneath. Multivariate analysis, adjusting for confounders, showed that a raised hut open underneath had 53% fewer mosquitoes (95% CI 47-58%), those with air-permeable walls underneath 24% fewer (95% CI 9-36%) and huts with solid walls underneath 31% fewer (95% CI 24-37%) compared with a hut on the ground. Similar results were found for Mansonia spp. and total number of female mosquitoes, but not for Culex mosquitoes where hut entry was unaffected by height or barriers. Indoor temperature and carbon dioxide levels were similar in all huts. CONCLUSION: Raising a house 2 m from the ground reduces the entry of An. gambiae and Mansonia mosquitoes, but not Culex species. The protective effect of height is reduced if the space underneath the hut is walled off.

Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species.

BACKGROUND: Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. METHODS: For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. RESULTS: Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. CONCLUSIONS: In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps.

A high heterozygosity genome assembly of Aedes albopictus enables the discovery of the association of PGANT3 with blood-feeding behavior.

The Asian tiger mosquito, Aedes albopictus, is a global invasive species, notorious for its role in transmitting dangerous human arboviruses such as dengue and Chikungunya. Although hematophagous behavior is repulsive, it is an effective strategy for mosquitoes like Aedes albopictus to transmit viruses, posing a significant risk to human health. However, the fragmented nature of the Ae. albopictus genome assembly has been a significant challenge, hindering in-depth biological and genetic studies of this mosquito. In this research, we have harnessed a variety of technologies and implemented a novel strategy to create a significantly improved genome assembly for Ae. albopictus, designated as AealbF3. This assembly boasts a completeness rate of up to 98.1%, and the duplication rate has been minimized to 1.2%. Furthermore, the fragmented contigs or scaffolds of AealbF3 have been organized into three distinct chromosomes, an arrangement corroborated through syntenic plot analysis, which compared the genetic structure of Ae. albopictus with that of Ae. aegypti. Additionally, the study has revealed a phylogenetic relationship suggesting that the PGANT3 gene is implicated in the hematophagous behavior of Ae. albopictus. This involvement was preliminarily substantiated through RNA interference (RNAi) techniques and behavioral experiment. In summary, the AealbF3 genome assembly will facilitate new biological insights and intervention strategies for combating this formidable vector of disease. The innovative assembly process employed in this study could also serve as a valuable template for the assembly of genomes in other insects characterized by high levels of heterozygosity.

Insights and challenges of insecticide resistance modelling in malaria vectors: a review.

BACKGROUND: Malaria is one of the most devastating tropical diseases, resulting in loss of lives each year, especially in children under the age of 5 years. Malaria burden, related deaths and stall in the progress against malaria transmission is evident, particularly in countries that have moderate or high malaria transmission. Hence, mitigating malaria spread requires information on the distribution of vectors and the drivers of insecticide resistance (IR). However, owing to the impracticality in establishing the critical need for real-world information at every location, modelling provides an informed best guess for such information. Therefore, this review examines the various methodologies used to model spatial, temporal and spatio-temporal patterns of IR within populations of malaria vectors, incorporating pest-biology parameters, adopted ecological principles, and the associated modelling challenges. METHODS: The review focused on the period ending March 2023 without imposing restrictions on the initial year of publication, and included articles sourced from PubMed, Web of Science, and Scopus. It was also limited to publications that deal with modelling of IR distribution across spatial and temporal dimensions and excluded articles solely focusing on insecticide susceptibility tests or articles not published in English. After rigorous selection, 33 articles met the review's elibility criteria and were subjected to full-text screening. RESULTS: Results show the popularity of Bayesian geostatistical approaches, and logistic and static models, with limited adoption of dynamic modelling approaches for spatial and temporal IR modelling. Furthermore, our review identifies the availability of surveillance data and scarcity of comprehensive information on the potential drivers of IR as major impediments to developing holistic models of IR evolution. CONCLUSIONS: The review notes that incorporating pest-biology parameters, and ecological principles into IR models, in tandem with fundamental ecological concepts, potentially offers crucial insights into the evolution of IR. The results extend our knowledge of IR models that provide potentially accurate results, which can be translated into policy recommendations to combat the challenge of IR in malaria control.

Detection of dengue in German tourists returning from Ibiza, Spain, related to an autochthonous outbreak, August to October 2022.

In February 2023, German public health authorities reported two dengue cases (one confirmed, one probable) and four possible cases who travelled to Ibiza, Spain, in late summer/autumn 2022; the infection was probably acquired through mosquito bites. Case 1 visited Ibiza over 1 week in late August with two familial companions; all three developed symptoms the day after returning home. Only Case 1 was tested; dengue virus (DENV) infection was confirmed by presence of NS1 antigen and IgM antibodies. Case 2 travelled to Ibiza with two familial companions for 1 week in early October, and stayed in the same town as Case 1. Case 2 showed symptoms on the day of return, and the familial companions 1 day before and 3 days after return; Case 2 tested positive for DENV IgM. The most probable source case had symptom onset in mid-August, and travelled to a dengue-endemic country prior to a stay in the same municipality of Ibiza for 20 days, until the end of August. Dengue diagnosis was probable based on positive DENV IgM. Aedes albopictus, a competent vector for dengue, has been present in Ibiza since 2014. This is the first report of a local dengue transmission event on Ibiza.

An optical system to detect, surveil, and kill flying insect vectors of human and crop pathogens.

Sustainable and effective means to control flying insect vectors are critically needed, especially with widespread insecticide resistance and global climate change. Understanding and controlling vectors requires accurate information about their movement and activity, which is often lacking. The Photonic Fence (PF) is an optical system that uses machine vision, infrared light, and lasers to identify, track, and interdict vectors in flight. The PF examines an insect's outline, flight speed, and other flight parameters and if these match those of a targeted vector species, then a low-power, retina-safe laser kills it. We report on proof-of-concept tests of a large, field-sized PF (30 mL × 3 mH) conducted with Aedes aegypti, a mosquito that transmits dangerous arboviruses, and Diaphorina citri, a psyllid which transmits the fatal huanglongbing disease of citrus. In tests with the laser engaged, < 1% and 3% of A. aegypti and D. citri, respectfully, were recovered versus a 38% and 19% recovery when the lacer was silenced. The PF tracked, but did not intercept the orchid bee, Euglossa dilemma. The system effectively intercepted flying vectors, but not bees, at a distance of 30 m, heralding the use of photonic energy, rather than chemicals, to control flying vectors.

Wolbachia-carrying Aedes mosquitoes for preventing dengue infection.

BACKGROUND: Dengue is a global health problem of high significance, with 3.9 billion people at risk of infection. The geographic expansion of dengue virus (DENV) infection has resulted in increased frequency and severity of the disease, and the number of deaths has increased in recent years. Wolbachia,an intracellular bacterial endosymbiont, has been under investigation for several years as a novel dengue-control strategy. Some dengue vectors (Aedes mosquitoes) can be transinfected with specific strains of Wolbachia, which decreases their fitness (ability to survive and mate) and their ability to reproduce, inhibiting the replication of dengue. Both laboratory and field studies have demonstrated the potential effect of Wolbachia deployments on reducing dengue transmission, and modelling studies have suggested that this may be a self-sustaining strategy for dengue prevention, although long-term effects are yet to be elucidated. OBJECTIVES: To assess the efficacy of Wolbachia-carrying Aedes speciesdeployments (specifically wMel-, wMelPop-, and wAlbB- strains of Wolbachia) for preventing dengue virus infection. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, four other databases, and two trial registries up to 24 January 2024. SELECTION CRITERIA: Randomized controlled trials (RCTs), including cluster-randomized controlled trials (cRCTs), conducted in dengue endemic or epidemic-prone settings were eligible. We sought studies that investigated the impact of Wolbachia-carrying Aedes deployments on epidemiological or entomological dengue-related outcomes, utilizing either the population replacement or population suppression strategy. DATA COLLECTION AND ANALYSIS: Two review authors independently selected eligible studies, extracted data, and assessed the risk of bias using the Cochrane RoB 2 tool. We used odds ratios (OR) with the corresponding 95% confidence intervals (CI) as the effect measure for dichotomous outcomes. For count/rate outcomes, we planned to use the rate ratio with 95% CI as the effect measure. We used adjusted measures of effect for cRCTs. We assessed the certainty of evidence using GRADE. MAIN RESULTS: One completed cRCT met our inclusion criteria, and we identified two further ongoing cRCTs. The included trial was conducted in an urban setting in Yogyakarta, Indonesia. It utilized a nested test-negative study design, whereby all participants aged three to 45 years who presented at healthcare centres with a fever were enrolled in the study provided they had resided in the study area for the previous 10 nights. The trial showed that wMel-Wolbachia infected Ae aegypti deployments probably reduce the odds of contracting virologically confirmed dengue by 77% (OR 0.23, 95% CI 0.15 to 0.35; 1 trial, 6306 participants; moderate-certainty evidence). The cluster-level prevalence of wMel Wolbachia-carrying mosquitoes remained high over two years in the intervention arm of the trial, reported as 95.8% (interquartile range 91.5 to 97.8) across 27 months in clusters receiving wMel-Wolbachia Ae aegypti deployments, but there were no reliable comparative data for this outcome. Other primary outcomes were the incidence of virologically confirmed dengue, the prevalence of dengue ribonucleic acid in the mosquito population, and mosquito density, but there were no data for these outcomes. Additionally, there were no data on adverse events. AUTHORS' CONCLUSIONS: The included trial demonstrates the potential significant impact of wMel-Wolbachia-carrying Ae aegypti mosquitoes on preventing dengue infection in an endemic setting, and supports evidence reported in non-randomized and uncontrolled studies. Further trials across a greater diversity of settings are required to confirm whether these findings apply to other locations and country settings, and greater reporting of acceptability and cost are important.

Morphometry of the wings of Anopheles aquasalis in simulated scenarios of climate change.

BACKGROUND: Climate change has significant implications on ecosystems. We verified the effects of climate change on the malaria vector Anopheles aquasalis using simulated climate change scenarios (SSCCs). METHODS: An experimental model was designed for SSCCs, which composed of air-conditioned 25 m3 rooms. RESULTS: The wing size was significantly different between SSCCs. A colony of Anopheles aquasalis could not be established in extreme scenarios. CONCLUSIONS: Increases in temperature and CO2 in the atmosphere may modify the global epidemiology of malaria, marking its emergence in currently malaria-free areas.

Laboratory evaluation of the contact irritancy of a clothianidin solo formulation vs. clothianidin-deltamethrin mixture formulations for indoor residual spraying against pyrethroid-resistant Anopheles gambiae sensu lato.

BACKGROUND: Clothianidin-based indoor residual spraying (IRS) formulations have become available for malaria control as either solo formulations of clothianidin or a mixture of clothianidin with the pyrethroid deltamethrin. While both formulations have been successfully used for malaria control, studies investigating the effect of the pyrethroid in IRS mixtures may help improve our understanding for development of future IRS products. It has been speculated that the irritant effect of the pyrethroid in the mixture formulation may result in shorter mosquito contact times with the treated walls potentially leading to a lower impact. METHODS: We compared contact irritancy expressed as the number of mosquito take-offs from cement surfaces treated with an IRS formulation containing clothianidin alone (SumiShield® 50WG) to clothianidin-deltamethrin mixture IRS formulations against pyrethroid-resistant Anopheles gambiae sensu lato under controlled laboratory conditions using a modified version of the World Health Organisation cone bioassay. To control for the pyrethroid, comparison was made with a deltamethrin-only formulation. Both commercial and generic non-commercial mixture formulations of clothianidin and deltamethrin were tested. RESULTS: The clothianidin solo formulation did not show significant contact irritancy relative to the untreated control (3.5 take-offs vs. 3.1 take-offs, p = 0.614) while all deltamethrin-containing IRS induced significant irritant effects. The number of take-offs compared to the clothianidin solo formulation (3.5) was significantly higher with the commercial clothianidin-deltamethrin mixture (6.1, p = 0.001), generic clothianidin-deltamethrin mixture (7.0, p < 0.001), and deltamethrin-only (8.2, p < 0.001) formulations. The commercial clothianidin-deltamethrin mixture induced similar contact irritancy as the generic clothianidin-deltamethrin mixture (6.1 take-offs vs. 7.0 take-offs, p = 0.263) and deltamethrin-only IRS (6.1 take-offs vs. 8.2, p = 0.071), showing that the irritant effect in the mixture was attributable to its deltamethrin component. CONCLUSIONS: This study provides evidence that the enhanced contact irritancy of the pyrethroid in clothianidin-deltamethrin IRS mixtures can shorten mosquito contact times with treated walls compared to the clothianidin solo formulation. Further trials are needed to directly compare the efficacy of these formulation types under field conditions and establish the impact of this enhanced contact irritancy on the performance of IRS mixture formulations containing pyrethroids.

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

Mapping malaria vectors and insecticide resistance in a high-endemic district of Haryana, India: implications for vector control strategies.

BACKGROUND: Achieving effective control and elimination of malaria in endemic regions necessitates a comprehensive understanding of local mosquito species responsible for malaria transmission and their susceptibility to insecticides. METHODS: The study was conducted in the highly malaria prone Ujina Primary Health Center of Nuh (Mewat) district of Haryana state of India. Monthly entomological surveys were carried out for adult mosquito collections via indoor resting collections, light trap collections, and pyrethrum spray collections. Larvae were also collected from different breeding sites prevalent in the region. Insecticide resistance bioassay, vector incrimination, blood meal analysis was done with the collected vector mosquitoes. RESULTS: A total of 34,974 adult Anopheles mosquitoes were caught during the survey period, out of which Anopheles subpictus was predominant (54.7%). Among vectors, Anopheles stephensi was predominant (15.5%) followed by Anopheles culicifacies (10.1%). The Human Blood Index (HBI) in the case of An. culicifacies and An. stephensi was 6.66 and 9.09, respectively. Vector incrimination results revealed Plasmodium vivax positivity rate of 1.6% for An. culicifacies. Both the vector species were found resistant to DDT, malathion and deltamethrin. CONCLUSION: The emergence of insecticide resistance in both vector species, compromises the effectiveness of commonly used public health insecticides. Consequently, the implementation of robust insecticide resistance management strategies becomes imperative. To effectively tackle the malaria transmission, a significant shift in vector control strategies is warranted, with careful consideration and adaptation to address specific challenges encountered in malaria elimination efforts.

Spatial analysis of dengue transmission in an endemic city in Brazil reveals high spatial structuring on local dengue transmission dynamics.

In the last decades, dengue has become one of the most widespread mosquito-borne arboviruses in the world, with an increasing incidence in tropical and temperate regions. The mosquito Aedes aegypti is the dengue primary vector and is more abundant in highly urbanized areas. Traditional vector control methods have showing limited efficacy in sustaining mosquito population at low levels to prevent dengue virus outbreaks. Considering disease transmission is not evenly distributed in the territory, one perspective to enhance vector control efficacy relies on identifying the areas that concentrate arbovirus transmission within an endemic city, i.e., the hotspots. Herein, we used a 13-month timescale during the SARS-Cov-2 pandemic and its forced reduction in human mobility and social isolation to investigate the spatiotemporal association between dengue transmission in children and entomological indexes based on adult Ae. aegypti trapping. Dengue cases and the indexes Trap Positive Index (TPI) and Adult Density Index (ADI) varied seasonally, as expected: more than 51% of cases were notified on the first 2 months of the study, and higher infestation was observed in warmer months. The Moran's Eigenvector Maps (MEM) and Generalized Linear Models (GLM) revealed a strong large-scale spatial structuring in the positive dengue cases, with an unexpected negative correlation between dengue transmission and ADI. Overall, the global model and the purely spatial model presented a better fit to data. Our results show high spatial structure and low correlation between entomological and epidemiological data in Foz do Iguaçu dengue transmission dynamics, suggesting the role of human mobility might be overestimated and that other factors not evaluated herein could be playing a significant role in governing dengue transmission.

Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Physical durability and insecticidal activity of long-lasting insecticidal nets in Cruzeiro do Sul, Brazil.

Vector control is one of the principal strategies used for reducing malaria transmission. Long-lasting insecticidal bed nets (LLINs) are a key tool used to protect populations at risk of malaria, since they provide both physical and chemical barriers to prevent human-vector contact. This study aimed to assess the physical durability and insecticidal efficacy of LLINs distributed in Cruzeiro do Sul (CZS), Brazil, after 4 years of use. A total of 3000 LLINs (PermaNet 2.0) were distributed in high malaria risk areas of CZS in 2007. After 4 years of use, 27 'rectangular' LLINs and 28 'conical' LLINs were randomly selected for analysis. The evaluation of physical integrity was based on counting the number of holes and measuring their size and location on the nets. Insecticidal efficacy was evaluated by cone bioassays, and the amount of residual insecticide remaining on the surface of the LLINs was estimated using a colorimetric method. After 4 years of use, physical damage was highly prevalent on the rectangular LLINs, with a total of 473 holes detected across the 27 nets. The upper portion of the side panels sustained the greatest damage in rectangular LLINs. The overall mosquito mortality by cone bioassay was < 80% in 25/27 rectangular LLINs, with panel A (at the end of the rectangular bednet) presenting the highest mortality (54%). The overall mean insecticide concentration was 0.5 µg/sample, with the bednet roof containing the highest average concentration (0.61 µg/sample). On the conical LLINs, 547 holes were detected, with the bottom areas sustaining the greatest damage. The cone bioassay mortality was < 80% in 26/28 of the conical LLINs. The mean insecticide concentration was 0.3 µg/sample. After 4 years of use, the insecticidal efficacy of the LLINs was diminished to below acceptable thresholds.